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PubMed1992

[Effects of fatty acids on hepatic amino acid metabolism in isolated perfused liver in rats].

Large disparity in experimental conditions concerning the use of isolated perfused liver led us to investigate the effects of two free-fatty acids on amino acid metabolism in this model. Oleate uptake was 210 +/- 46 nmol/min.g and octanoate uptake 550 +/- 60 nmol/min.g. Fatty acid utilization led to an increase in ketogenesis, enhanced by octanoate in accordance with its known availability for beta-oxidation. In our experiments neither oleate nor octanoate modified hepatic amino acid exchange and metabolism as assessed by the measurements of amino acids fluxes and glucose and urea production.

Amino Acids↗
PubMed1986

Pharmacokinetics of melphalan in isolated perfusion of the limbs.

The pharmacokinetics of melphalan (L-phenylalanine mustard) in isolated perfusion treatment of patients with melanoma in the limbs have been studied at standardized pseudo-physiological perfusion conditions. Perfused tissue volumes ranged from 2.1 to 16 liters as measured by water displacement. A fixed dose of 10 mg of the drug per liter of perfused tissue was applied. The resulting variation in total melphalan dosage gave rise to varying drug concentrations in the perfusate as the extracorporeal system was operated with a fixed volume of priming fluid. Perfusion with melphalan was applied for 60 min. Concentrations of intact drug were assayed by high-performance liquid chromatography. The melphalan concentrations vs. time for each patient were fitted to a biexponential equation using a nonlinear least-squares computer program. Mean half-lives of 4.7 +/- 0.3 and 53.0 +/- 1.6 min were obtained for the alpha and beta phase, respectively. The hybrid constant alpha remained virtually constant with increasing dose (i.e., increasing drug concentration) and beta also appeared independent of dose levels. With increasing tissue volume plasma clearance was found to diminish per unit of tissue volume. This phenomenon, and a levelling off of the steady-state distribution volume with increasing volume of perfused tissue, are discussed in terms of a possibly restricted transfer of drug from intravascular to the extracellular space and of the possibility of saturation of cellular uptake systems in the bulk of the limb tissues.

Adult↗
PubMedDate not supplied

Absence of metabolism of morphine during accumulation by isolated perfused rabbit lung.

Pulmonary accumulation and metabolism of morphine was studied in an isolated perfused lung system. No metabolism of morphine or N-demethylation of ethylmorphine was found. Accumulation from the recirculating perfusate was nonsaturable over a concentration range of 3-400 microM morphine. Inhibition of sodium transport or sodium depletion decreased morphine accumulation. The results are most consistent with passive diffusion of morphine down an electrochemical gradient and subsequent nonspecific binding to tissue anions. Normal movement of sodium and potassium is required to maintain this gradient.

Animals↗
PubMedDate not supplied

Uptake and disposition of chlorinated biphenyls by isolated perfused rat liver.

The hepatic disposition of four chlorinated biphenyls was studied in isolated perfused rat liver preparations perfused with 30% rat blood. Biliary excretion of 14C-labeled 4-chlorobiphenyl (1-CB): 4,4'-dichlorobiphenyl (2-CB): 2,4,5,2',5'-pentachlorobiphenyl (5-CB) and 2,4,5,2',5'-hexachlorobiphenyl (6-CB) appeared to be inversely related to increased chlorination. Each of the compounds was rapidly taken up by the liver from the circulating perfusate. After 4 hr of perfusion, biliary excretion of 14C from 1-CB, 2-CB, 5-CB, and 6-CB was 48.2, 29.6, 20.5, and 1.3% of total dose, respectively. For 1-CB, this rate of biliary elimination represents a maximum perfusate/bile ratio of 107. About 97% of the label in the bile from 1-CB experiments was in the form of metabolites. Biliary excretion of 1-CB was biphasic: an initial rapid phase (t 1/2 = 13 min) was followed by a slower second phase (t 1/2 = 122 min). When the metabolites of 1-CB were added to the perfusate, biliary excretion of these metabolites was monophasic; t 1/2 = 120 min, which corresponded to the slower phase observed when the parent compound was presented to the liver. 1-CB disappeared from the perfusate via a biphasic process. Metabolites of 1-CB effused into the perfusate immediately after the initial rapid uptake of 1-CB by the liver, and the recirculating pool of metabolites was eliminated in the bile by the slow postsynthetic phase of biliary elimination. Less than 2% of 1-CB was present as the parent compound in the perfusion system after 4 hr of perfusion. These findings indicate that the metabolism of 1-CB is not the limiting factor in the hepatic disposition of 1-CB and that the circulating metabolites of 1-CB effusing from the liver might be the major source for urinary excretion.

Animals↗
PubMed1976

[Insulin and glucagon secretion in the isolated perfused rat pancreas].

For the study of insulin and glucagon secretion at the isolated perfused rat pancreas are used, as a rule, synthetic perfusion media that have to meet two requirements: They have to ensure the biological function of the perfused organ for 2 hrs, and must not interfere with radioimmunological insulin and glucagon determination. An appropriate medium is the Krebs-Ringer bicarbonate buffer at pH 7.4 with addition of 4% dextran (MG 75000) and 0.5% human serum albumin; it allows one to produce, after permanent glucose stimulation, a biphasic insulin secretion. Infusion media based on gelatine or polyvinylpyrrolidone proved inadequate for these studies.

Animals↗
PubMed2006

Effects of genetic deletion of angiotensin-(1-7) receptor Mas on cardiac function during ischemia/reperfusion in the isolated perfused mouse heart.

In this study we investigated the role of Mas on cardiac function during ischemia/reperfusion in isolated perfused mouse heart. Following a stabilization period of 30 min, hearts from WT and Mas KO mice were subjected to global ischemia. After 20 min of ischemia, the flow was restarted and the hearts were reperfused for 30 min. An additional group of WT mice was perfused with solution containing the Ang-(1-7) receptor Mas antagonist A-779. Isolated heart of Mas KO and WT treated with A-779 presented an increase in the perfusion pressure in the baseline period. This difference increased with 5 min of reperfusion reaching similar values to baseline period at the end of the reperfusion. Isolated hearts of Mas KO and WT treated with A-779 also presented a decreased systolic tension, +/-dT/dt, and HR. Upon global ischemia WT hearts showed a significant decrease in systolic tension and an increase in diastolic tension. During reperfusion an increase in systolic and diastolic tension was observed in WT mice. Deletion or blockade of Mas markedly attenuated these changes in isolated hearts. These results indicate that Mas plays an important role in cardiac function during ischemia/reperfusion which is in keeping with the cardiac and coronary effects previously described for Ang-(1-7).

Angiotensin I↗
PubMed1979

Effects of glucose and parathyroid hormone on the renal handling of myoinositol by isolated perfused dog kidneys.

The effects of glucose and parathyroid hormone (PTH) on the transport and metabolism of myoinositol (MI) and [2-(3)H]MI were studied in isolated perfused dog kidneys. Studies during perfusion of kidneys with normal and elevated glucose concentrations demonstrated that under normal conditions the isolated kidney reabsorbed 94.7+/-0.2% of the filtered MI, and the renal production of (3)H-metabolities of MI was 117.9+/-6% of the filtered MI load. This indicated that entry of MI into tubular cells by reabsorption was not the sole pathway for entry into the pool of MI within the kidney undergoing catabolism. High glucose perfusate decreased MI reabsorption to 68.6+/-4.7% and thus decreased delivery of [2-(3)H]MI into the catabolic pool from the reabsorptive pathway. In the high glucose experiments, the rate of [2-(3)H]MI catabolism exceeded [2-(3)H]MI reabsorption by the same fraction as in normal glucose experiments, which indicates that high glucose did not affect nonreabsorptive access of MI to the catabolic site. In contrast to the effects of glucose, PTH administration resulted in an increase in perfusate MI concentration and a decrease in the perfusate [2-(3)H]MI specific activity. Concomitantly, urinary MI and [2-(3)H]MI concentrations were increased, again with a decrease in [2-(3)H]MI specific activity. These results indicate that PTH caused a release of MI into the urine (not the same as decreased MI reabsorption, which would not affect urinary [(3)H]MI specific activity) and into the perfusate of the isolated kidneys. These effects on MI release were about coincidental with the increase in urinary cyclic 3',5'-AMP after PTH and preceded the peak phosphaturic effect of PTH. There was no detectable effect of PTH on MI synthesis from glucose as a source of the MI released into the urine and perfusate. However, PTH temporarily halted accumulation of tritiated MI catabolites. There was no effect of inactivated PTH on urinary cyclic 3',5'-AMP or on MI transport, which indicates that the PTH effect on MI handling was a specific hormonal effect. These studies clarify the renal metabolism of MI, and they demonstrate heretofore unknown effects of PTH on the renal handling and metabolism of MI. The effects of PTH on renal MI metabolism have important implications in renal carbohydrate metabolism and phospholipid turnover.

Animals↗
PubMed1983

Variations in cellular attenuation and vesicle numerical densities in capillary endothelium and type I epithelium of isolated, perfused dog lungs after acute severe edema formation.

Morphometric comparisons of nonedematous and edematous isolated, perfused dog lungs establish that there are significant differences between the degree of cellular attenuation and vesicle numerical densities in endothelial and type I epithelial cells of the alveolar septa after edema production. In nonedematous isolated lungs the extent of endothelial and epithelial attenuation was greater on the thin sides of the septa. In the edematous lungs, the differential of greater thin-side attenuation was maintained for the endothelium but not for the epithelium where the extent of attenuation in the septal thick segments was increased. Vesicle numerical densities were approximately doubled in the cells on both sides of the septa in the edematous lungs. The endothelial vesicle densities were greater in the septal thin segments than in the septal thick segments in both the nonedematous and the edematous isolated lungs. The epithelial vesicle densities, on the other hand, were similar on the thin and thick sides of the septa in the nonedematous and edematous lung preparations. Although the contribution of vesicles to cellular function in the alveolar septa remains uncertain, further evaluation of vesicular transport should include the possible variability of this function with the varying degrees of cellular attenuation on the two anatomically distinct sides of the septa.

Animals↗
PubMed1981

Adrenergic and cholinergic regulation of lung surfactant secretion in the isolated perfused rat lung and in the alveolar type II cell in culture.

The purpose of this study is to define the hormonal regulation of pulmonary surfactant secretion in two models, the isolated perfused rat lung and the isolated alveolar type II cell in culture. In the perfused lung, both cholinergic and adrenergic stimulation independently increased labeled disaturated phosphatidylcholine secretion, the major phospholipid component of surfactant, each by 2.3-fold. A concomitant increase in lung cGMP and cAMP concentration of 275- and 25-fold, respectively, was observed. The effect of each agonist was inhibited only by its appropriate antagonist. In alveolar type II cells in culture, both adenylate and guanylate cyclase responded to their appropriate agonists and antagonists. The release of 3H-labeled disaturated phosphatidylcholine was enhanced by beta-adrenergic but not alpha-adrenergic or cholinergic agonists. The effect of isoproterenol (10 microM) on surfactant release was seen by 2.5 min, and secretion was stimulated 2.9-fold at a half-maximal concentration of 1 nM. Cyclic AMP levels were increased by 4.9-fold by isoproterenol at a half-maximal concentration of 40 nM. These results indicate that while in the perfused lung, secretion is stimulated by both adrenergic and cholinergic effectors, in the type II cell model, surfactant secretion is under only beta-adrenergic control.

Acetylcholine↗
PubMed1990

Metabolic activation and bronchiolar Clara cell necrosis from naphthalene in the isolated perfused mouse lung.

A method for isolation and maintenance of the mouse lung ex vivo suitable for the study of the relationship between metabolism and toxicity is described. Physical, biochemical and morphological evaluations revealed that the lung is viable for up to 5 hr. No significant alterations in the architecture of the bronchiolar epithelium were observed by light or electron microscopy, despite evidence of interstitial and peribronchial edema. Although increases in pulmonary arterial pressure were noted at 5 hr, lung wet/dry weight was elevated only minimally (11%). Glutathione levels remained stable for the first 3 hr but fell to 57 +/- 14% of control at 5 hr. Naphthalene monooxygenase activity was not altered significantly during 5 hr of perfusion. Perfusion of the lung with naphthalene resulted in swelling and vacuolation of Clara cells followed by concentration-dependent losses of this cell type from the bronchiolar epithelium. Clara cells comprised 63% of the epithelial cells in terminal airways of control mice; 10 mumol of naphthalene decreased this number to 30%. Perfusion with naphthalene resulted in concentration-dependent decreases in pulmonary glutathione. Reactive metabolites were bound covalently to protein in the lung and in the perfusate. This study is the first to provide unambiguous evidence that naphthalene-induced Clara cell necrosis can be mediated entirely by processes resident in the lung. In addition, this work validates the use of the isolated perfused mouse lung for studying the role of reactive metabolites produced in situ vs. those entering the lung via the circulation.

Animals↗
PubMed1982

The molecular nature of vascularly released cholecystokinin from the isolated perfused porcine duodenum.

Using sequence-specific radioimmunoassays, the quantities and molecular nature of cholecystokinin (CCK) have been determined in extracts of porcine duodenal mucosa and in the vascular perfusate from the isolated porcine duodenum. The basal concentration of CCK in the perfusate was 84 pM equiv. CCK-8 (mean; range: 32-173 pM, n = 5). After intraluminal stimulation with amino acids, acidified fat emulsions and hydrochloric acid, the concentrations increased 2--5-fold. Both in the basal and stimulated state the concentrations of the related hormone, gastrin, were below 5 pM equiv. gastrin-17. CCK in the perfusate was concentrated by affinity-chromatography using antibodies directed against the bioactive C-terminus. Subsequent gel chromatography revealed a form with a size like or slightly larger than the C-terminal dodecapeptide (CCK-12), a predominant form resembling the C-terminal octapeptide (CCK-8), and a form resembling the C terminal tetrapeptide (CCK-4). The duodenal mucosa contained in addition CCK-33, -39 and CCK-peptides with further N-terminal extensions. The results suggest that small CCK peptides are the principal circulating forms, while CCK-33 and larger forms are biosynthetic precursors.

Amino Acid Sequence↗
PubMed1979

Excretion and metabolism of catecholamines by the isolated perfused rat kidney.

The excretion and metabolism of labeled epinephrine and norepinephrine by the isolated, perfused rat kidney were studied. The excretion of both catecholamines significantly exceeded the amount filtered, thus providing direct evidence of net tubular secretion. Renal clearance of epinephrine was significantly greater than that of norepinephrine. Tubular secretion was a linear function of the concentration of unbound catecholamine in the medium with no demonstrable tubular maximum at the concentrations studied. The isolated kidney removed catecholamines from the medium by metabolism as well as excretion in the urine. O-Methylation was the major metabolic route and O-methylated metabolites were rapidly excreted and concentrated in urine. Preferential excretion and metabolism of epinephrine were confirmed in double-label experiments in which [14C]epinephrine and [3H]norepinephrine were perfused together. The ratio of 14C:3H in urine exceeded that in perfusion medium for total radioactivity as well as for catecholamines and O-methylated amines. The present study thus provides direct evidence for (a) net tubular secretion of epinephrine and norepinephrine with a direct relationship between secretion and medium concentration; (b) significant renal metabolism of both epinephrine and norepinephrine with O-methylation as the major metabolic route; and (c) preferential excretion and metabolism of epinephrine.

Animals↗
PubMed1992

The effects of isoproterenol on the isolated perfused rat heart.

In isolated hearts infused with up to 100 ug/ml isoproterenol (ISO), there was only a transient effect on the frequency of contraction, and no effect on buffer flow through the heart. Infusion of hearts with 40-60 or 70-100 ug/ml ISO produced an increase in the release of lactate dehydrogenase, but not creatine kinase. Microscopic examination of hearts revealed myocyte separation and cytoplasmic vacuolation, of similar severity, in both control and ISO infused hearts; there was no necrosis. The data from this experiment are consistent with the view that ISO has little or no direct toxic effect on the heart. Since ISO is cardiotoxic In vivo, it is reasonable to suppose that this toxicity is linked primarily to the pharmacological effects of the drug. However, our study does not exclude the possibility that under the stressed conditions prevailing in vivo following a challenge with ISO, the susceptibility of myocytes to a direct toxic effect of ISO may increase.

Animals↗
PubMed1984

Application of the isolated perfused rat liver preparation to pharmacological studies of the intrahepatic portal vascular bed.

A method is described for the perfusion of the isolated rat liver preparation which is particularly suited to a selective pharmacological study of the portal vascular bed of the liver. A synthetic medium free of vasoactive substances and composed of 2.5% bovine serum albumin in Krebs-Henseleit buffer is pumped into the portal vein at a controlled rate while the perfusion pressure is recorded electronically. The advantages of this technique are that while the viability of the isolated organ is preserved, the preparation remains haemodynamically stable and the response to vasoactive agents can be accurately and reproducibly determined. The constant perfusate flow also allows cumulative dose-response curves to be constructed, even for powerful vasoconstrictor agents. The present perfusion method has also been validated by comparing the responses obtained from a variety of vasoactive substances with those reported in other in vivo studies.

Acetylcholine↗
PubMed1987

Evaluation of potential heart affine compounds by use of the isolated perfused rat heart.

99mTc-organocation complexes were screened for heart affinity using the isolated perfused rat heart. The 99mTc-I compounds are better extracted by the heart and retained in it than the 99mTc-III compounds. 99mTcCl2(DMPE)2+ shows low uptake (15%) and fast washout, but no impairment of uptake after incubation with human serum albumin(HSA), with a modified preparation, initial uptake is not much better (18%) but washout is clearly diminished. 99mTc(DMPE)3+ is taken up to a greater extent (33%) if administered solely, but when given in conjunction with HSA, uptake decreases to about 4% because of strong affinity of the complex to the protein, but this compound persists well in the tissue. Detergents like tween 80 and dioctylsulphosuccinate are able to cause higher uptake of 99mTc(DMPE)3+ even in the presence of HSA, thus indicating a diminished binding of the compound to the protein. 99mTc(CN-t-butyl)6+ shows very high uptake (80%) by the heart, which is only moderately diminished by addition of HSA (to 38%), and persists excellently in the myocardium. Basic features of the compounds such as accumulation in and elimination from the myocardium as well as plasma protein binding and their mutual relationships are well reflected by the isolated heart model.

Animals↗